Calmodulin (CaM) is a ubiquitous calcium (Ca) binding protein which bind myosin light chain kinase (MLCK), caldesmon (CaD), calponin (Calp) and a sarcolemmal Ca-ATPase to regulate the contraction of vascular smooth muscle. Ca binding to CaM stimulates it binding to each of these proteins but little is known concerning the rates at which Ca-CaM binds and dissociates from these important target proteins during a Ca transient. We will use fluorescence stopped-flow with our fluorescent CaM's to determine the Kd's and the rates of association and dissociation of each of these CaM-target protein complexes and determine the rate at which each complex is disrupted by Ca removal. We will determine if these target proteins have differential effects on Ca exchange rates with CaM's N- and C-terminal Ca binding sites and relate Ca dissociation from these sites to the rate of EGTA induced complex disruption. We will study naturally occurring mutant CaM's to determine if altered Ca binding to their N- and C-terminal Ca binding sites is responsible for their inability to activate Na and K ion channels, respectively. This should yield important new information on the mechanism by which Ca-CaM binds and activates more than 20 different cellular target proteins. Hormonal activation of the Ca phospholipid dependent protein kinase C (PKC) results in its activation at resting Ca levels and its phosphorylation o MLCK, CaD, Calp, and Ca-ATPase to modulate and maintain smooth muscle contraction. We will determine if PKC phosphorylation of these target proteins alters CaM banding and activation. We have, for the first time directly measured Ca off-rates from purified PKC and the Ca-ATPase. This will allow us to determine the mechanism and extent that physiological activators of these two enzymes modulate their Ca affinity and their activity. These studies should increase our understanding of how hormonal activation modulates these Ca binding proteins and CaM's interaction with its target proteins in smooth muscle.